PCR conditions | 16S (625 bp) | blaTEM (618 bp) | nim (458 bp) | |
Initial denaturation | 95°C for 5 min | 94°C for 5 min | 94°C for 10 min | |
Denaturation | 94°C for 1 min | 94°C for 1 min | 94°C for 30 s | |
Annealing | 55°C for 1 min | 62°C for 1 min | 62°C for 1 min | |
Extension | 72°C for 1 min | 72°C for 1 min | 72°C for 1 min | |
Step 2 to 4 | 34 cycles | 35 cycles | 32 cycles | |
Final extension | 72°C for 5 min | 72°C for 10 min | 72°C for 10 min | |
DNA template | 5 µl | 5 µl | 5 µl | |
dNTPs mix, 10 mM | 1 µl (200 µM) | 2 µl (400 µM) | 1 µl (200 µM) | |
Reaction Buffer 5x | 10 µl | 10 µl | 10 µl | |
MgCl2 Solution, 25 mM | 3 µl (1.5 mM) | 4 µl (2 mM) | 3 µl (1.5 mM) | |
Upstream Primer, 10 µM | 2.5 µl (0.5 µM) | 5 µl (1 µM) | 5 µl (1 µM) | |
Downstream Primer, 10 µM | 2.5 µl (0.5 µM) | 5 µl (1 µM) | 5 µl (1 µM) | |
Taq polymerase, 5 U/µl | 0.25 µl (1.25 U) | 0.25 µl (1.25 U) | 0.25 µl (1.25 U) | |
Water for injection | 25.75 µl | 18.75 µl | 20.75 µl | |
Values in parenthesis are final concentration |